Methods
We conducted a secondary analysis of the Stillbirth Collaborative Research Network (SCRN) study, a racially/ethnically diverse, population-based case-control study of stillbirth with enrollment at the time of delivery. The SCRN study recruitment sites and study population details have been previously described.4, 12 The study was supported by grant funding from the Eunice Kennedy ShriverNational Institute of Child Health and Human Development: U10-HD045953 Brown University, Rhode Island; U10-HD045925 Emory University, Georgia; U10-HD045952 University of Texas Medical Branch at Galveston, Texas; U10-HDO45955 University of Texas Health Sciences Center at San Antonio, Texas; U10-HD045944 University of Utah Health Sciences Center, Utah; and U01-HD045954 RTI International, RTP. Secondary analysis of the primary research was supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number 1UL01TR002538. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. An advisory board reviewed the progress and safety of the study and written informed consent was obtained from each participant. Study participants were not involved in the development of the research and a core outcome set has not been used in the research.
In the present analysis, we excluded cases with multifetal gestations and without perinatal postmortem examination data. Among eligible cases, we included those with and without major fetal anomalies as described previously.13 Women were enrolled at the time of diagnosis of stillbirth, diagnosed as fetal death that occurred at 20 weeks’ gestation or more. Gestational age was determined by the best clinical estimate from multiple sources including information from assisted reproductive technology, last menstrual period and/or obstetric ultrasound.14 Women underwent a standardized maternal interview, medical record abstraction, biospecimen collection, an obstetric ultrasound exam, and postmortem examinations of the fetus and placenta. The standardized postmortem protocol ensured evaluation of stillbirth consistently across study geographic regions to best identify stillbirth where a fetal or placental condition caused or significantly contributed to the fetal death.15 To avoid inadvertent exclusion of fetuses that may have been ≥20 weeks of gestation, fetal deaths occurring at 18-19 weeks and 6 days estimated gestational age with poor gestational dating criteria were included as stillbirth cases.12, 13 Fetal autopsy and placental examinations of stillbirth cases were performed by perinatal pathologists using study-specific standardized protocols, that included centralized training.15, 16
Fetal structural malformations were prenatally diagnosed and grouped as major malformations by anatomic system and specific malformation type as described previously.13 The groupings include cystic hygroma, central nervous system (open neural tube defect, anencephaly, hydranencephaly, hydrocephalus, holoprosencephaly, other) using a protocol for neuropathologic examination,16 thoracic (congenital diaphragmatic hernia, cystic adenomatoid malformation, pulmonary sequestration, other), cardiac (anteroseptal defect, ventricular septal defect, atrioventricular canal defect, transposition of the great vessels, tetralogy of Fallot, other), gastrointestinal (gastroschisis, omphalocele, duodenal atresia, other), genitourinary (hydronephrosis/ureteropelvic junction obstruction, autosomal recessive polycystic kidney disease, multicystic/dysplastic kidney, posterior urethral valves, renal agenesis, other), skeletal (skeletal dysplasia, club feet, other), umbilical cord, craniofacial (cleft palate, other), hydrops fetalis, other and any anomalies. To further describe malformations, a ‘write in’ option was used, where sections were individually reviewed to ensure appropriate categorization. For example, ‘craniofacial anomalies’ category was added after data inspection revealed that these were not consistently categorized. In addition, a single diagnosis for ‘hydrops’ category was added, and when multiple abnormalities were noted and consistent with hydrops (i.e. ascites, pleural effusions, skin oedema). Other structural malformations were defined based on ‘write in’ option, where sections were individually reviewed to ensure that they were appropriately categorized. Multiple structural malformations (any malformation) were classified according to each system for which an abnormality was present.
Placental and stored/frozen fetal liver, fetal muscle and cord blood tissue biospecimens were collected from stillbirth cases. Sizes of placental biopsies varied, but they were as large as 1 cm3.16 In macerated fetuses, fresh samples were obtained from the placenta. DNA from placental biopsies was stored at −20°C for 2 to 5 years before microarray analysis, which was performed at a single laboratory (Columbia University Medical Center). DNA from stored frozen muscle and liver specimens was extracted immediately before microarray analysis and were used when placental DNA was unavailable for analysis. Fetal muscle, cord blood or fetal liver were used for chromosomal microarray assessment when placental DNA was unavailable (n=106 [19.9%]).
DNA Samples were analyzed using the Affymetrix Genome Wide Human single nucleotide polymorphism (SNP) Array 6.0 and the Chromosome Analysis Suite, version 1.0.1, and the NetAffx annotation database, version 28 for microarray analysis (Affymetrix). Data were aligned to the Human Genome release 18 (hg18). CNVs with ≥500 kb in size were detected using the SNP array. Analysis of the array data was conducted to determine aneuploidy, potential maternal-fetal contamination, and sex discordance. Classification of CNVs was based on the American College of Medical Genetics (ACMG) standards and guidelines for interpretation and reporting, with modifications as described previously.4, 17 Due to improving resolution for determination of pathogenicity of CNVs, the number of novel structural variants is constantly increasing.18 An efficient computational analysis may be required to update pathogenicity score of manually defined variants of unknown clinical significance (VOUS) CNVs. Therefore, we implemented the latest ACMG guidelines19in high-throughput CNV analysis to classify and update pathogenicity of CNVs previously categorized as VOUS by using ClassifyCNV tool.20 Using the ACMG guideline, we classified CNVs into two groups: abnormal CNVs, defined as pathogenic CNVs (including aneuploidy) or VOUS, and normal CNVs, defined as no CNVs > 500 kb or benign CNVs.21-23 As such, the abnormal CNVs and normal CNVs groups were compared in statistical analysis and only pathogenic CNVs (excluding aneuploidy, sex-chromosome and VOUS CNVs) were discussed.
We used the Wald Chi-squared test and a two-by-two table to compare the proportions of abnormal CNVs and normal CNVs between stillborn fetuses with and without fetal structural malformations. Other categorical measures were similarly compared between abnormal CNVs and normal CNVs. To compare continuous measures, ANOVA statistics was used. Data were analyzed with the use of statistical software programs: SAS version 9.4 (SAS Institute Inc), R and STATA version 15.0 (StataCorp), and ClassifyCNV tool.20